Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells

Abstract

Nitric oxide in the absence of oxygen was suggested to react with 5–50 mM glutathione (GSH) over many minutes when [NO ] ≪ [GSH] (N. Hogg et al., FEBS Lett. 382:223–228; 1996). However, Aravindakumar et al. ( J. Chem. Soc. Perkin Trans. 2:663–669; 2002) provided data suggesting ∼200-fold higher reactivity under conditions of [NO ] ≫ [GSH]. To help resolve these differences, the rate of loss of NO (∼9 μM) in aqueous solutions of GSH (2.5–20 mM) was measured by chemiluminescence. An apparent second-order rate constant of 0.080 ± 0.008 M −1 s −1 at pH 7.4, 37°C, was calculated based on the total [GSH] and “pseudo-first-order” kinetics; thiolate anion was much more reactive than undissociated thiol. These data imply a half-life of ∼30 min for low concentrations of NO with 5 mM GSH, 37°C, pH 7.4, in the absence of oxygen. Possible kinetic schemes that can partially explain the divergent literature reports are discussed, notably an equilibrium in the reaction between NO and GSH. Human breast carcinoma MCF-7 cells were exposed to NO (initially ∼18 μM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in ∼60 min. Aerobic exposure depletes GSH in cells in vitro much faster because of autoxidation of NO to NO 2 , >10 8 times more reactive toward GSH.